This pilot, multicenter, randomized, controlled study was carried out in accordance with the recommendations of the “Ethical guidelines for medical and health research involving human subject, Ministry of Education, Culture, Sports, Science, and Technology in Japan,” with written informed consent from all subjects. All study protocols were approved by the institutional review boards of each participating facility. The study was registered with the University Hospital Medical Information Network Clinical Trial Registry (UMIN000020927), which is a public trial registry.

Patients were recruited from outpatient stroke clinics affiliated with three participating facilities. The inclusion criteria were as follows: age, 20–90 years; and with a first stroke in chronic stage (>180 days from stroke onset). The exclusion criteria were as follows: bilateral or brain stem infarct or hemorrhage; voluntary extension of the metacarpophalangeal and interphalangeal joints of three or more fingers ≤10° or voluntary wrist extension ≤20°; severe impairment in balance or walking, indicated by the need for assistance for standing, walking or using the toilet; substantial use of the affected upper extremity before the intervention, indicated by a score of >2.5 points on the amount of use (AOU) scale of the Motor Activity Log (MAL); clear signs of dementia or cognitive disorder, indicated by a score <24 points in the Mini-Mental State Examination; severe aphasia or apraxia, preventing the patient from participating in the activities involved in the study intervention; presence of another uncontrolled medical condition or severe end-stage disease; and severe contraction in the area of the shoulder, elbow, wrist, or fingers.

Before initiating the trial, we calculated that ten patients with chronic stroke should be enrolled in each group. A previous study suggested that a pilot study sample size should be 10% of sample size of project study (30). The largest CIMT study conducted to date was the EXCITE study (3), which enrolled about 100 patients in each group. Additionally, Hwetzog (31) recommended that pilot studies include 10–40 patients per group to provide an accurate estimation of treatment outcome. Therefore, we estimated 10 patients in each group would be sufficient.

Participants were randomized into the control group (conventional CIMT) or the treatment group (dual-tDCS and PNMES plus CIMT) by a researcher blinded to group allocation and who was not directly involved in this study. Randomization was performed according to Zelen’s method, combined with minimization algorithm to control for the following factors: age (in years), time from stroke onset (in days), and baseline upper extremity score based on Fugl-Meyer Assessment (FMA) (32).

The patients in the treatment group first received dual-tDCS for 20 min, followed immediately by PNMES, and then CIMT for 2 h. Pretreatment stimulation (dual-tDCS and PNMES) was applied both before the morning CIMT session and before the afternoon CIMT session (Figure 1). Dual-tDCS was performed using a DC-STIMULATOR PLUS (neuroConn GmbH, Ilmenau, Germany). For dual-tDCS, the anode was placed over the affected primary motor cortex (point C3 or C4 according to the 10–20 system), while the cathode was placed over the unaffected primary motor cortex (point C4 or C3 according to the 10–20 system). The following stimulation protocol was used for tDCS: constant current of 1-mA intensity (15), applied for 20 min (33), followed by PNMES performed using a TORIO stimulation system (Ito Co. Ltd., Tokyo, Japan), with two self-adhesive electrodes placed on the extensor digitorum muscle. Trains of electrical stimulation (20 Hz, on/off duty cycle 150/150μs; pulse duration, 300 µs) were applied at 1 Hz for 10 min. Stimulation intensity was set at a level where each patient reported mild paresthesia, but no pain, and minimal visible muscle contractions were evoked (23). Patients in the control group did not receive any stimulation. Sham stimulation was not used because the PNMES device used in this study did not have a setting permitting sham stimulation.

All patients received 4 h of CIMT (2 h in the morning and 2 h in the afternoon) from Monday to Friday for 2 weeks (10 consecutive weekdays). CIMT was provided by trained occupational therapists, according to a CIMT protocol described in detail elsewhere (34). CIMT is based on three main principles, namely: repetitive task-oriented training (shaping and task practice); training to facilitate the transfer of functional gains achieved in the clinical setting to the activities of daily living in real life (“transfer-package” training); and restraint of the less affected upper extremity using a mitt.

We assessed the motor function of the affected upper extremity and its use in real-world behaviors, before and immediately after CIMT, using the FMA for the upper extremity, primary outcome (32), and MAL [AOU and quality of movement (QOM)], as the secondary outcome, respectively (35). The FMA for the upper extremity consists of 33 items, each scored on a 3-point ordinal scale: 0 point, cannot perform the action; 1 point, can perform the action only partially; and 2 points, can perform the action fully. Thus, the maximum possible upper extremity score was 66 points. The AOU and QOM scores, components of the MAL, indicate how much and how well, respectively, the affected upper extremity is used during 14 activities of daily living. For each activity of daily living, the patient rates the extent of the activity performed and how well it can be performed using the affected upper extremity. MAL scoring uses a 6-point Likert scale, ranging from 0 (never used) to 5 (used as prior to stroke). The mean AOU score reflects the frequency of the activity, whereas the mean QOM score reflects how well the activity was performed. The maximum value for the AOU and QOM scores is 5 points. The FMA and MAL assessments were performed by blinded and trained occupational therapists who was not directly involved in the treatment or patient allocation to intervention groups.

All data were analyzed using JMP version 13.0 (SAS Institute, Cary, NC, USA). Between-group differences in baseline characteristics were assessed using Fisher’s exact test (categorical data) or unpaired t test (ordinal data). Treatment effectiveness between the treatment and control group, which was assessed by analysis of covariance (ANCOVA), which was used to control for the baseline FMA score, and the AOU and QOM score of MAL, respectively. Finally, differences in upper extremity motor function, between baseline and postintervention, were assessed within each group using paired t-test. In all statistical comparisons, a P < 0.05 was considered statistically significant. The effect size index η² (ANCOVA) and Δ (paired t-test) were also calculated. Data were presented as the mean (SD).

Several researchers have defined the minimum clinically important difference regarding paretic upper extremity motor recovery in chronic stroke patients as an improvement above 4.25 points in the FMA upper extremity score and above 0.5 points in the AOU score (36, 37). In our study, both groups achieved improvements higher than these thresholds of clinically important difference on the FMA and AOU. Additionally, on the FMA upper extremity scale, the improvement was 4.64 points higher in the treatment than in the control group, and this between-group difference was also above the threshold for a clinically meaningful change. These findings suggest that behavioral motor treatment, combined with tDCS and PNMES, can provide meaningful improvement in upper limb function chronic stroke patients. On other hand, we found no significant between-group difference between in terms of the QOM score of the MAL. However, η² of the effect size of the between-group difference was 0.43. According to Cohen, effect size assessed in terms of η² is considered small effect for η² < 0.01, medium effect for 0.01 < η² < 0.06, and large effect for η² > 0.14 (38). Therefore, although the P-value (P = 0.07) did not indicate a significant difference between the groups in terms of the QOM score of MAL, the η² value indicated that this difference was indicative of a substantial effect of the combined treatment. This discrepancy might be explained by statistical errors (type II error or false negatives), most likely related to the small sample size and thus insufficient power in the statistical analysis. Additionally, Lang et al. (39) reported that the minimum clinically important difference regarding the improvement in the QOM score of MAL in stroke patients amounts to an increase of 1.00–1.10 points. In our study, the minimum clinically important difference in the QOM score of MAL was noted in treatment group but not in the control group, which supports the conclusion that there is consistently higher improvement in the treatment group even though the between-group difference in QOM score improvement was not statistically significant.

The novelty of the present study lies in that we used a combination of dual-tDCS and PNMES, rather than only dual-tDCS or PNMES, to prolong the duration of the neural modulation effect. We found a clinically meaningful improvement in the motor function of the affected limb, reflected in the 9.2-point improvement in FMA upper extremity score [from 43.00 (9.82) to 52.20 (8.28)]. A previous study reported that dual-tDCS followed by CIMT achieved only a 6.3-point improvement in FMA upper extremity score (from 25.4 to 31.7 points) (22). This previous study used a similar CIMT treatment protocol as that applied in the present study (4 h per day for 10 consecutive weekdays). Compared to these previous observations, our findings indicated higher improvement in motor function on the affected side, although it should be noted that the degree of severity of deficit at baseline differed substantially between the two studies (patients in the present study were less affected upper extremity function compared to patients of the previous study). Therefore, our result showed that the novelty combination stimulation (dual-tDCS combined with the PNMES) strategy holds promise to improve the affected motor function rather than the dual-tDCS stimulation strategy alone. However, in this study, we could not make a strong claim about effectiveness of our novelty stimulation strategy, because we did not directly compare between the above two different stimulation strategies.

The limitations of our study should be noted. First, the sample size of the present study was very small, with 10 patients in the treatment group and 9 patients in control group. We are planning to perform future trials after performing power calculations to estimate the minimum sample size necessary to ascertain the effectiveness of the combined treatment. The results obtained in this pilot study will serve for calculating the expected effect size. Second, we did not include a sham group because the device for PNMES did not have a setting for sham stimulation. Our result might include the placebo effect in which patients in the treatment group received some positive psychological effects because they understood that the PNMES might provide a positive treatment effect. Therefore, the placebo effect acting on the data regarding patients in the treatment group cannot be excluded. Third, in this study, we established the group that received CIMT alone as a control group. However, to investigate the effects of dual-tDCS combined with PNMES, we should establish a group that received CIMT combined with the dual-tDCS. We plan to address this issue in a further study.

Despite the above limitations, the present trial clearly suggested that, compared to behavioral and motor rehabilitation alone, non-invasive stimulation with dual-tDCS and PNMES followed by behavioral and motor treatment provides greater effectiveness to enhance the recovery of motor function and real-world use of the affected upper extremity in patients with chronic stroke. Therefore, in chronic stroke patients, the novel pretreatment based on dual-tDCS and PNMES may enhance the therapeutic benefit of CIMT.